Dephosphorylation and quantification of organic ... - naldc - USDA

Chemosphere 72 (2008) 1782–1787

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Dephosphorylation and quantification of organic phosphorus in poultry litter by purified phytic-acid high affinity Aspergillus phosphohydrolases Thanh H. Dao a,*, Khanh Q. Hoang b a b

USDA, ARS, Environmental Management and ByProducts Utilization Laboratory, BARC-East, Bldg. 306 Powder Mill Road, Beltsville, MD 20705, USA Applied Microbiology Laboratory, Institute of Tropical Biology, Viet Nam Academy of Science and Technology, Ho Chi Minh City, Viet Nam

a r t i c l e

i n f o

Article history: Received 9 January 2008 Received in revised form 12 April 2008 Accepted 14 April 2008 Available online 13 June 2008 KeyWords: Extracellular enzymes Enzymatic dephosphorylation Poultry litter Phytate Phosphorus Waste management

a b s t r a c t Extracellular phosphohydrolases mediate the dephosphorylation of phosphoesters and influence bioavailability and loss of agricultural P to the environment to pose risks of impairment of sensitive aquatic ecosystems. Induction and culture of five strains of Aspergillus were conducted to develop a source of high-affinity and robust phosphohydrolases for detecting environmental P and quantifying bioactive P pools in heterogeneous environmental specimens. Enzyme stability and activity against organic P in poultry litter were evaluated in 71 samples collected across poultry producing regions of Arkansas, Maryland, and Oklahoma of the US Differences existed in strains’ adaptability to fermentation medium as they showed a wide range of phytate-degrading activity. Phosphohydrolases from Aspergillus ficuum had highest activity when the strain was cultured on a primarily chemical medium, compared to Aspergillus oryzae which preferred a wheat bran-based organic medium. Kinetics parameters of A. ficuum enzymes (Km = 210 lM; Vmax of 407 nmol s1) indicated phytic acid-degrading potential equivalent to that of commercial preparations. Purified A. ficuum phosphohydrolases effectively quantified litter bioactive P pools, showing that organic P occurred at an average of 54 (±14)% of total P, compared to inorganic phosphates, which averaged 41 (±12)%. Litter management and land application options must consider the high water-extractable and organic P concentrations and the biological availability of the organic enzymelabile P pool. Robustness of A. ficuum enzymes and simplicity of the in situ ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to sense for on-farm accumulation of such environmentally-sensitive P forms. Published by Elsevier Ltd.

1. Introduction Extracellular phosphohydrolases have received increased interest as dietary supplements in rations of monogastric livestock in recent years (Boling et al., 2000; CAST, 2002). The practice was mandated by state legislative action to protect water quality and aquatic ecological systems of sensitive estuarine watersheds such as the Chesapeake Bay in the US by nutrient loss from intensive animal production agriculture. Poultry and livestock are fed highenergy grain-based rations which contain high levels of phytic acid (myo-inositol hexakisphosphate). These monogastric or singlestomach species cannot utilize P in phytic acid because they do not possess sufficient levels of phytic acid-degrading enzymes in their digestive system to break down this organic source of P (CAST, 2002). Similarly, high concentrations of dietary polyvalent cations such as Ca or Al, also inhibited organic P dephosphorylation and resulted in ruminants (Bos taurus) excreta having significant

* Corresponding author. Tel.: +1 301 504 8315. E-mail address: [email protected] (T.H. Dao). 0045-6535/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.chemosphere.2008.04.048

levels of phosphohydrolase-labile P (Dao, 2003; Toor et al., 2005; Dao et al., 2006) and in runoff from manure-amended soils (Green et al., 2007). In these studies, the researchers used phosphomonesterases to quantify the enzyme-labile P fraction, which indicated that the organic P substrates were potentially bioavailable phosphomonoesters. Although phytic acid and other phosphomonoesters can be quantified by 31P nuclear-magnetic resonance spectroscopy, mass spectrometry, or liquid chromatography, these techniques are not conducive to routine analysis of heterogeneous multi-phase environmental samples such as manures, soils, or natural water and sediments. Applications of spectrometric and enzymatic methods and their limitations have been extensively addressed elsewhere (Dao, 2006). Because of elaborate sample preparation and specialized instrumentation, spectroscopic analyses require a large investment of time and labor that contribute to the high cost of an analysis. A simple enzymatic fractionation assay was developed in our laboratory, readily providing evidence that large pools of inorganic and organic phosphohydrolase-labile P forms existed in manure of dairy cattle (Dao et al., 2006), and in other grain-fed livestock species (CAST, 2002; He et al., 2006; Dao et al., 2007). Furthermore, the determination of the availability

T.H. Dao, K.Q. Hoang / Chemosphere 72 (2008) 1782–1787

and biological significance of these P pools may well be equally or more important than the chemical identification of specific monoesters to the management and development of mitigation practices to reduce phosphate release and delivery to the environment. The dephosphorylation of inositol phosphates, in particular phytic acid, is carried out by extracellular phosphohydrolases, and often called phytases or phytic acid-degrading enzymes. The complete dephosphorylation of phytic acid yields six mol of orthophosphate and one mol of myo-inositol. Histidine acid- and alkaline phosphohydrolases are produced by a wide variety of organisms, including bacteria (i.e., Bacillus subtilis), yeasts (i.e., Sacharomyces), and fungi (i.e., Aspergillus) (Mullaney et al., 2000; Vohra and Satyanarayana, 2003). These enzymes are produced as animal feed supplements, and extensive research efforts have been devoted to the improvement of enzyme performance at stomach and gut pH, their thermal stability and substrate specificity (Vats et al., 2005; Weaver et al., 2007). Extracellular phosphohydrolases are recently thought as a means of detecting environmental P accumulation in soil and animal manure and assessing risks of water quality impairment or eutrophication (Turner et al., 2002; Dao, 2003, 2004; Vats et al., 2005; Green et al., 2007). Enzymatic methods have been applied to the identification of organic P-containing substrates in soil extracts, runoff, and surface water columns but have not been used as quantitative methods for soil P pools because of their low efficiency when compared to spectrometric techniques (Turner et al., 2002; Dao, 2003; He et al., 2006). Improvements have recently been made to in situ enzymatic methods by enhancing substrate availability and therefore quantitative recovery of enzyme-labile P content of samples of manure, soils, or runoff (Dao, 2003, 2004, 2006; He et al., 2006; Green et al., 2007). Dao (2003) first reported the approach of using polycarboxylate ligands in combination with extracellular enzymes to simultaneously extract, hydrolyze, and categorize P species, without separate steps of an extraction and isolation of the extract for enzymatic assay. The hydrolysis reaction is strongly driven to reach an equilibrium state toward the dephosphorylation of organic P. The rationale and mechanisms for enhanced efficiency were extensively discussed in previous work (Dao, 2003, 2006). The in situ experimental approach was distinctly different from other extraction and enzymatic methods (e.g., Hedley et al., 1982; Turner et al, 2002; He et al., 2006). In the two-step approach, the extraction of solid-phase P might not be complete or exhaustive and the extraction process was terminated upon separation and removal of the liquid phase for analysis of extracted P. Factors that affected analytical efficiency included the extractant’s strength (i.e., organic acids vs. strong mineral acids or bases), the harshness of extraction conditions (i.e., mild vs. vigorous agitation), and the length of extraction time prior to the determination of P in the extract or the addition of enzymes to the extract and subsequent analysis of extracted organic P. Simplicity, accuracy, and high throughput of an enzymatic technique are also needed to reliably measure reactive P species in environmental samples. For such a purpose, extracellular enzymes that are stable, have high substrate affinity, and can retain efficacy in heterogeneous multi-phase environmental samples such as manure, natural waters, and sediments are critically needed. Culture media and growth conditions have been observed to influence growth and enzyme induction in A. niger (Papagianni et al., 1999, 2001). Natural variability also exists in Aspergillus strains in their ability to produce phosphohydrolases and in the enzymes’ specific activity. Novel high-producing strains and high affinity phosphohydrolases offer the potential for enhanced feed P assimilation in the animal as dietary supplements, in addition to applications in environmental P sensing. Although major advances have been made in the understanding of the biogeochemistry of inorganic and organic forms in the last two decades, particularly on inositol

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phosphates, knowledge gaps still exist in the understanding of factors influencing their speciation, biological availability, and release mechanisms in highly heterogeneous manure solids such as poultry litter, and other by-products of animal feeding operations (Zhang et al., 2002; Dao and Zhang, 2007). The scarcity of information on the stability of these organic forms in manure, their mineralization, and release of inorganic P present a major challenge to understanding environmental P’s fate. This study was conducted to (1) determine the effects of submerged culture conditions on the growth and induction of extracellular phosphohydrolases in five selected Aspergillus strains, (2) characterize the kinetic rate of phytic acid degradation by the most active enzymes and (3) determine the phosphohydrolases’ stability and catalytic potential by applying the purified enzymes to the fractionation of bioactive P pools in poultry litter. 2. Materials and methods 2.1. Aspergillus culture conditions Five sources of phosphohydrolase-producing Aspergillus that included Aspergillus ficuum (Reichert) Hennings, anamorph (ATCC 66876) and Aspergillus oryzae strains isolated from rice (Oryza sativa L.) fermentation pomace (i.e., AMR1, AMR2, and AMR5) and ATCC 96684. The ATCC strains were obtained from the American Type Culture Collection (Manassas, Va.). All sources were maintained on potato dextrose agar (PDA) consisting of potato infusion (250 g l1), dextrose (10 g l1), and agar (15 g l1). The final pH was adjusted to 5.5. The culture was maintained by periodic transfer on PDA slants and stored at 4 °C. Induction and production of fungal phosphohydrolases were carried out by inoculating 1 cm2 of Aspergillus spore grown on PDA amended with penicillin-streptomycin (1%) in corn starch-peptone based media (Papagianni et al., 1999). In summary, Medium 1 (in g l1) contained corn starch (28), peptone (18), glucose (5), KCl (0.5), Mg(SO4) (1.5), KH2PO4 (1), and CaCl2 (2). Medium 2 contained wheat bran (20) and had reduced concentration of starch (2.8), glucose (0.5) and peptone (1.8). The culture flask was incubated at 37 °C on an orbital shaker at 300 rpm for up to 7d. The culture medium for each of the fungal strains was filtered and the crude filtrate free of fungal colonyforming units (CFU) containing the phosphohydrolases was used to determine enzymatic activity, pH, and total extracellular protein content by a dye-binding method (Bradford, 1976). 2.2. Enzyme purification and concentration 2.2.1. Acetone precipitation and lypholyzation Aliquots (100 ml) of the crude CFU-free filtrates were vigorously mixed (150 rpm) with an equal volume of acetone. After decanting the supernatant that contained most of the culture media components and metabolic byproducts, the precipitated protein fraction was washed with additional acetone (10 ml) and the solid residue was re-dissolved in a known volume of deionized water (5 ml). One-ml aliquot of the purified enzyme solution was assayed for phytate-degrading potential. 2.2.2. Ultracentrifugation Purified enzymes were also prepared by re-filtering the crude CFU-free filtrates through a polyether sulfone filter membrane with a molecular weight cut-off of 30,000 (Sartorius, Edgewood, NY) by ultracentrifugation. The fraction >30 kDa was assayed for phytate-degrading potential in triplicate 2-ml aliquots and the results were compared to those of the precipitation–lypholyzation procedure to detect any potential detrimental effect of acetone precipitation on enzyme activity.

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2.3. Phytate-degrading enzymatic activity assay Phytate-degrading activity in crude CFU-free filtrates and in preparations of enzymes purified by ultracentrifugation and by lypholyzation was determined by measuring the release of inorganic P from a standard solution of 1.0 mM phytic acid (Dao, 2003). Twenty-five millilitres of crude CFU-free filtrates or subsamples of ultracentrifuged or lypholyzed enzymes dissolved in 25 ml of deionized water were incubated at 37 °C with 1.0 mM sodium phytate stock solution (pH 4.3) in a total volume of 100 ml. Duplicate 1-ml aliquots were periodically removed (0, 15, 30, 60 min, etc. up to 6 h), immersed in boiling water to stop the dephosphorylation reaction before P analysis. Phosphate-P concentrations released over time of incubation were determined by the phosphomolybdate-ascorbic acid method using an autoanalyzer (APHA, 1998). The first-order kinetic rate model was used to describe phosphate-P concentration distributions to determine and compare reaction rate coefficients and specific activity of enzyme preparations from the five strains of Aspergillus. In addition, a commercial source of enzymes was used as a reference, for comparing phytic acid-degrading activities of in-house enzyme preparations. A. ficuum phosphohydrolases purified by acetone precipitation– lypholyzation, henceforth referred to as purified phosphohydrolases, were used to determine enzyme kinetic characteristics and bioactive P fractions in poultry litter. Kinetic parameters Vmax and Km were calculated from the Lineweaver–Burk enzyme-catalyzed reaction equation.

1 Km 1 1 ¼ ; þ V i V max ½PA V max

dry matter content following drying at 65 °C to express all extracted P concentrations on a dry weight basis. Elemental composition of powdered litter samples was also determined by polarized x-ray fluorescence (XRF) spectroscopy (Dao and Zhang, 2007). Calcium, Mg, Al, Fe, Mn, Cu, P, and As concentrations were quantified on an XRF spectrometer (model XLAB2000, Spectro-USA, Fitchburg, MA). In addition, total P and N concentrations of acid-digests, using a concentrated H2SO4-persulfate mixture were also determined using the phosphomolybdate-ascorbic acid and the indophenol blue methods, respectively. 2.5. Statistical analysis The procedure PROC CLUSTER was applied to segregate the litter samples into clusters of related P and Ca concentrations (SAS, 2004). Binary relationships between P fractions and selected manure characteristics were fitted to linear models using PROC GLM. Comparisons of Lineweaver–Burk plots or other multiple linear regression curves were made using proc GLM and a CLASS statement as outlined in Dao et al. (1982). 3. Results 3.1. Effect of growth medium on the induction and activity of Aspergillus extracellular phosphohydrolases Differences existed in adaptability of Aspergillus strains to the media used for submerged fermentation (Fig. 1). The five Aspergillus

where Vmax is the reaction maximal velocity, expressed as nmol s1 (i.e., nanokatal) and Km is a measure of substrate affinity of the phosphohydrolases. 2.4. Poultry litter bioactive P fractionation Bulk samples (ca 2 kg) of manure cake and litter scrapped from the floor of poultry production houses during periodic clean up, and feed samples were collected from farms located on the Eastern Shore of Maryland and throughout the western Arkansas-eastern Oklahoma poultry producing areas (n = 71). Bioactive P fractions in poultry litter samples were determined according to the procedure developed by Dao (2003, 2006, Dao et al., 2006) where four fractions were differentiated. They included water-extractable phosphate-P (WEP), ligand-exchangeable inorganic phosphate-P (EEPi), ligand-exchangeable organic phosphohydrolase-labile P (EDTA-PHP), and the all-inclusive total bioactive P (WEP + EEPi + EDTA-PHP), which were determined as follows. The WEP fraction was determined in litter-water suspensions (1:100, w/v) in polycarbonate jars containing 0.1-g of powdered litter and deionized water. The suspensions were agitated at 250 rpm for 1 h, centrifuged at 10,000g for 15 min, and the supernatant was analyzed for phosphate-P concentrations. Aliquots of a 5 mM EDTA stock solution were added to another 0.1-g sample of litter (1:100, w/v) to determine the EEPi fraction. The mixtures were agitated at 250 rpm for 1 h. Aliquots of the solution-phase were centrifuged at 10,000g, and soon thereafter, the supernatant phosphate-P concentrations were determined. An aliquot (0.05 unit ml1) of purified A. ficuum was added to replace the volume removed for EEPi measurements and the litter-enzyme mixtures were equilibrated for 24 h. Aliquots (5 ml) of the supernatant were heated (100 °C) to stop the enzymatic reaction, then centrifuged at 10,000g, and the supernatant was analyzed for phosphate-P concentrations to determine the total bioactive P and calculate the EDTA-PHP fraction. Litter samples were air-dried and subsamples (10 g) were used to determine

Fig. 1. Effect of culture medium composition on phytic acid degradation by phosphohydrolases from five strains of Aspergillus, at 37 °C and an initial phytic acid concentration Co = 1 mM.

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strains showed a wide range in phytate-degrading activity. The highest phytic acid-degrading activity was obtained when A. ficuum was grown in the medium M1. Enzymes isolated from A. oryzae strains showed phytic acid-degrading activity but at a much reduced efficiency when the organisms were raised on the organic medium M2 containing wheat bran complex cell-wall polysaccharides and starch as sources of phytic acid. Crude CFU-free filtrates of the growth medium for the A. ficuum strain showed phytic acid-degrading potential equivalent to a commercial enzyme preparation (Fig. 2). Increasing the amounts of the crude filtrates of A. ficuum growth medium also increased the rate of dephosphorylation of the substrate and the eventual completion of the reaction by 24 h after filtrate addition (Fig. 2). Purification of the crude CFU-free filtrates by ultracentrifugation or protein precipitation-lypholyzation concentrated the enzymes and enhanced the specific activity of the resulting preparation under the assay conditions (i.e., Co = 1 mM, pH 4.3, 37 °C) (data not shown). 3.2. Enzyme kinetic characteristics The Km and Vmax parameters for the in-house purified A. ficuum phosphohydrolases and commercial enzymes are shown in Table 1, based on an equivalent unit of activity. Comparisons of Lineweaver–Burk regression curves for the enzymes showed only significant differences in intercepts (K 1 m ) between the in-house preparation and the commercial one, and the in-house phosphohydrolases showed a lower affinity for the substrate than the commercial enzymes. However, as a catalyst, both enzyme preparations effected the complete dephosphorylation of phytic acid, in overnight incubation at 37 °C (Fig. 2). The kinetic parameters were within range of or exceeded reported values for wildtype A. ficuum or niger strains (Casey and Walsh, 2003; Vats et al., 2005; Weaver et al., 2007). 3.3. Aspergillus ficuum phosphohydrolases and bioactive P forms in poultry litter As polyvalent cations concentrations in the litter may influence organic P mineralization and extraction of the released phosphate, two independent clusters were identified based on total Ca concentrations. Hierarchical cluster analysis was performed using the centroid method. Characteristics of the litter clusters are shown in Table 2. For reasons outlined in previous studies, the litter samples were collected from broiler (Cluster 1) and egg-pro-

Fig. 2. Rate of phytic acid degradation by commercial and in-house Aspergillus ficuum phosphohydrolases at enzyme concentrations ranging from 1 to 2-fold that of the commercial preparation (0.05 unit ml1) and 37 °C.

Table 1 Kinetic characteristics of phosphohydrolases from in-house submerged fermentation and a commercial source Enzyme source

Vmaxa (nmol s1)

Kmb (lM)

Commercial source Aspergillus ficuum in-house preparation

468 (±28) 407 (±10)

98 (±2) 210 (±5)

a b

Reaction maximal velocity (± standard error). Measure of substrate affinity of the phosphohydrolases.

Table 2 Pattern of clustering of 71 samples of poultry litter collected across poultry-producing regions of Maryland and Oklahoma, based on their calcium concentrationsa Clusterb

No. elements

Cluster mean (mmol kg1)

Distance between cluster centroids (mmol kg1)

1 2

64 7

0.95 3.04

2.09 2.09

a Determined by energy-dispersive X-ray fluorescence in powdered litter samples. b Method = centroid.

ducing (Cluster 2) operations. Calcium concentrations were 3fold higher (i.e., 91 ± 9 g kg1) in egg-laying poultry litter than broiler litter (Dao and Zhang, 2007). Broiler litter samples also contained significantly higher as concentration (i.e., 18-fold) than egglaying poultry litter samples, confirming the presence of two types of poultry manure in the set of litter samples. The purified A. ficuum phosphohydrolases were effective in quantifying the bioactive P pools in both types of litter samples in spite of their difference in Ca concentrations (Fig. 3). Overall, the WEP fraction and total bioactive (WEP + EEPi + EDTA-PHP) P fractions were proportional to the total P content of the litter samples, representing about 24% and most of the TP of the litter samples, respectively (Fig. 4). The high correlation between complexed inorganic and organic (EEPi + EDTA-PHP) pools and TP suggested that most of the P in the litter was complexed with polyvalent counterions and was exchangeable with EDTA and similar ligands. In addition, the TP concentration distribution suggested that approximately one-fifth of the litter samples had elevated TP levels that exceeded 21 g kg1 and WEP concentrations P5 g kg1. From a typical dietary concentration of 3–4 g P kg1, the variability in litter management practices across farms and producing regions resulted in highly enriched and variable WEP and bioactive P composition.

Fig. 3. Linear correlations between total P and water-extractable (WEP) and insoluble complexed inorganic and phosphohydrolase-labile P (EEPi + EDTA-PHP) fractions in poultry litter collected from poultry-producing regions of Arkansas, Maryland, and Oklahoma.

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Fig. 4. Distribution of bioactive forms of phosphorus relative to total phosphorus (TP) content in 71 samples of poultry litter collected across poultry-producing regions of Arkansas, Maryland, and Oklahoma, as determined by in-house Aspergillus ficuum phosphohydrolases and the ligand-based enzyme-labile assay.

Fig. 5. Relationship between total bioactive P (WEP + EEPi + EDTA-PHP) forms and total Ca or the sum of Ca and Mg concentrations in poultry litter collected from poultry-producing regions of Arkansas, Maryland, and Oklahoma.

The all-inclusive (WEP + EEPi + EDTA-PHP) fraction showed a high correlation with Ca concentration or slightly higher correlation to the sum of Ca and Mg concentrations, however only within the subset of broiler litter (Fig. 5); the above relationship felt apart in the egg-laying poultry litter group because of an excess of Ca that is needed in diets of egg-laying poultry for eggshell formation, and where the Ca:P ratio can be as high as 12:1 (NRC, 1994). No significant relationship was observed between any of the P pool and total Al or Fe or the sum of molar concentrations of these cations, partly due to the dominant presence of Ca in this type of manures. 4. Discussion In the wheat bran-amended culture medium 2, A. oryzae appeared less susceptible to the inorganic phosphate concentration in the chemical medium M1 (i.e., 230 mg l1). The phytic acidhydrolytic activity of A. oryzae phosphohydrolases was nevertheless low, compared to those of the A. ficuum strain. Other culture

conditions will have to be further developed to improve enzyme induction in A. oryzae, such as additional temperature, pH, and co-factors in future studies. The in-house preparation of phosphohydrolases induced the complete dephosphorylation of phytic acid, just as the commercial preparation did. The dephosphorylation of phytic acid appeared to be non-specific; once cleavage of the first ester C–O–P bond was initiated, the enzymes appeared to break any of the remaining ester bonds with equal ease because the overall rate of release of inorganic phosphate closely followed first-order reaction kinetics as shown in Fig. 1. A strong possibility exists that the organism for the two enzyme sources may belong to the same genus and species, i.e., A. ficuum. Commercial phosphohydrolase production has largely focused on A. niger which has been extensively used in the production of organic acids and many other enzymes. As the classification of Aspergillus is primarily based on morphological features, rather than biochemical or genetic characteristics, A. ficuum and A. niger are physically very similar. Therefore, the name A. niger often has been used interchangeably with A. ficuum (Oh et al., 2004; Vats et al., 2005; Ward et al, 2006). In this study, the source of the organism is A. ficuum and the kinetic results may well bear out the biochemical similarities in the two enzyme sources. Litter Ca concentrations played a dominant role in controlling solubility and susceptibility of organic P to dephosphorylation by phosphohydrolases. Coupled with the low water solubility of Ca salts of these organic phosphates, any enzymatic method would be hampered by low efficacy when used to determine P fractions in manure or in soils. Dao (2003, 2004) demonstrated the necessity of using low dosages of EDTA or similar polydentate ligands in enzymatic assays for determining bioactive P pools. Ligands increase the exchange and solubilization of complexed inorganic and organic P forms, and together with phosphohydrolases, will induce the hydrolysis and determination of soluble and complexed organic P forms. In the current study, the high Ca content did not appear to hamper the determination of bioactive P forms in the two subsets of litter samples. As a percentage of TP, the two clusters showed identical recovery rate although these two types of production systems greatly differ in ration composition and feeding regimes, as well as in their manure management practices. Organic phosphomonoesters or organic enzyme-labile P occurred in significant concentrations in poultry litter at current diet formulation and feeding regimes practiced on the farm in the Maryland Eastern Shore and the Arkansas–Oklahoma region of the US (Fig. 4). Because of the inorganic–organic P composition of poultry litter, there are serious implications for current litter management practices and land-based disposal options. While improved feed P assimilation in the animal remains a research and development priority, the reduction in dietary P in feed to essential levels must continue to decrease P excretion. These organic species are very dynamic and potentially biologically active in the environment, particularly in a carbon-rich environment. Previous research has shown internal rearrangement and inter-pool transfers over time that affected the mineralization of the enzyme-labile organic P pool (Dao, 2004; Dao et al., 2005). Spatial and temporal variability also exist in manure collected across a single farm (i.e., changes in diet composition because of growth stage-feeding practices); variations in nutrient assimilation efficiency over the animal life cycle, and in facility management practices affecting manure composition (i.e., collection and storage practices) also contribute to the dynamic composition of manure within a livestock production operation. The variability in P composition between samples and sampling locations also highlighted the difficulties in predicting P and other manure nutrients mineralization, and therefore, crop responses to manure when compared to commercial fertilizers. Therefore, a large number of samples and frequent analysis are necessary to accurately quantify and characterize these heterogeneous


manures (Dao et al., 2006; Dao and Zhang, 2007). The robustness of this source of A. ficuum phosphohydrolases and the simplicity of the ligand-based enzyme assay may thus increase routine assessment of litter bioactive P composition to detect and evaluate onfarm accumulation of such biologically active P forms. Timely knowledge of litter mineral composition may assist in efforts of maintaining optimal manure nutrient balance on-farm and on the production fields. The mild conditions of the in situ fractionation of litter P likely would not alter the chemical integrity of extracted P forms, avoiding chemical shifts between P pools and transformations that often accompany strongly acidic and/or basic chemical extractants. An enzyme-based assay also reveals the biological availability of the source P forms. Aspergillus and extracellular phosphohydrolases are ubiquitous in the environment, and with native phosphohydrolases of manure, and in soil and aquatic ecosystems, these enzymes increase the opportunities for hydrolysis of organic P and potentially induce P enrichment and favorable conditions for impairment of receiving waters. 5. Conclusions Extracellular phosphohydrolases are used as dietary supplements in rations of monogastric livestock to improve the assimilation of dietary P in the animal and reduce excretion in manure. Extracellular phosphohydrolases are also valuable biological tools for detecting loading and accumulation of P in animal manure, soil, and aquatic environments. The stability of purified enzymes and their hydrolytic activity against organic P in poultry litter were observed in A. ficuum and A. oryzea. High-affinity phosphohydrolases were produced locally for conducting the fractionation of P in poultry litter or other organic solids. The protocols for enzymes’ induction and purification can serve as standard procedures for evaluating novel sources of phosphohydrolases for biosensing organic P in the environment. The phosphohydrolases were effective in quantifying bioactive P pools and showed that on actual producers’ farms, poultry litter has over 50% of their TP as organic P forms. Manure management practices must take into account the bioavailability and time dependence of the mineralization of this substantial organic pool. Acknowledgements The authors sincerely acknowledged the technical assistance of G. Stone in the conduct of this study. The partial financial support provided by USDA-FAS under Agreement 60-1265-6004 to support Dr. K.Q. Hoang’s sabbatical in Beltsville, MD is sincerely acknowledged. References APHA, 1998. Phosphorus: automated ascorbic acid reduction method. In: Clescerl, L.S., Greenberg, A.E., Eaton, A.D., (Eds.), Standard Methods for the Examination of Water and Wastewater. Section 4500-P F, 20th ed. American Public Health Association, Washington, DC. Boling, S.D., Douglas, M.W., Shirley, R.B., Parsons, C.M., Koelkebeck, K.W., 2000. The effect of various levels of phytase and available phosphorus on performance of laying hens. Poultry Sci. 79, 535–538. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 162, 248–254.

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